DE10109690A1 - New nucleotide sequences encoding the metY gene - Google Patents

Abstract

The invention relates to an isolated polynucleotide comprising a polynucleotide sequence chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70 % to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70 % to the amino acid sequence of SEQ ID No. 2, c) polynucleotide which is complementary to the polynucleotides of a) or b), and d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c), and a process for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the metY gene coding for O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) is present in enhanced form, and the use of the polynucleotides which comprise the polynucleotide sequences according to the invention as hybridization probes.

Description

The invention relates to coding for the metY gene Nucleotide sequences from coryneform bacteria and a Process for the fermentative production of amino acids, in particular L-lysine and L-methionine, using Bacteria in which at least the metY gene is enhanced.

State of the art

L-amino acids, especially L-lysine and L-methionine find in human medicine and in the pharmaceutical industry, in the food industry and especially in the food industry Animal nutrition, application.

It is known that amino acids by fermentation of Strains coryneformer bacteria, in particular Corynebacterium glutamicum. Because of the Great importance is constantly attached to the improvement of Manufacturing process worked. process improvements can fermentation measures such as Stirring and supply of oxygen, or the Composition of nutrient media such as the Sugar concentration during fermentation, or the Work-up to the product form by, for example Ion exchange chromatography or the intrinsic Performance characteristics of the microorganism itself affect.

To improve the performance characteristics of this Microorganisms become methods of mutagenesis, selection and mutant selection applied. In this way you get Strains that are resistant to antimetabolites or auxotrophs for regulatory metabolites are and Produce amino acids.

For some years now also methods of recombinant DNA technology for strain improvement of L-  Amino acid producing strains of Corynebacterium Used by single amino acid biosynthetic genes amplified and the effect on the amino acid Production examined.

Object of the invention

The inventors have set themselves the task of new Measures for the improved fermentative production of Amino acids, in particular L-lysine and L-methionine, provide.

Description of the invention

Are mentioned in the following L-amino acids or amino acids, are thus including one or more amino acids their salts selected from the group L-asparagine, L- Threonine, L-serine, L-glutamate, L-glycine, L-alanine, L- Cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L- Tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine.

If in the following L-lysine or lysine are mentioned, are so that not only the bases, but also the salts such. B. Lysine monohydrochloride or lysine sulfate.

Are mentioned below L-methionine or methionine, are thus the salts such. For example, methionine hydrochloride or methionine sulphate.

The invention relates to an isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence coding for the metY gene selected from the group

• a) polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide, the the amino acid sequence of SEQ ID no. 2 contains  
• b) polynucleotide encoding a polypeptide having a Contains at least 70% of the amino acid sequence is identical to the amino acid sequence of SEQ ID NO. 2,
• c) polynucleotide that is complementary to Polynucleotides of a) or b), and
• d) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence from a), b) or c),

wherein the polypeptide preferably the activity of the O- Has acetyl homoserine sulfhydrylase.

The invention likewise provides the abovementioned polynucleotide, which is preferably a replicable DNA comprising:

• a) the nucleotide sequence shown in SEQ ID no. 1, or
• b) at least one sequence corresponding to the sequence (i) within the range of degeneration of the corresponds to genetic codes, or
• c) at least one sequence identical to that for the sequence (i) or (ii) complementary sequence hybridized, and optionally
• d) functionally neutral sense mutations in (i).

Other items are
a polynucleotide containing the nucleotide sequence as in SEQ ID no. 1 shown;
a polynucleotide encoding a polypeptide having the amino acid sequence as set forth in SEQ. ID. 2, contains;
a vector containing the coding for the metY gene DNA sequence of C. glutamicum, deposited under the Budapest Treaty in Corynebacterium glutamicum as pCREmetY on 13 05 00 under DSM 13556
and coryneform bacteria in which the metY gene is enhanced, in particular by the vector pCREmetY.

The invention also relates to polynucleotides which are in consist essentially of a polynucleotide sequence, the are available by screening by hybridization a corresponding gene bank of a coryneform bacterium, which contains the complete gene or parts thereof, with a probe containing the sequence of the invention Polynucleotide according to SEQ ID no. 1 or a fragment thereof contains and isolation of said polynucleotide sequence.

Polynucleotides containing the sequences according to the invention are included as hybridization probes for RNA, cDNA and DNA suitable for nucleic acids or polynucleotides or To isolate full-length genes for O-genes Acetyl homoserine sulfhydrolase or such To isolate nucleic acids or polynucleotides or genes, the high similarity of the sequence with that of the O- Acetyl homoserine sulfhydrolase gene.

Polynucleotides containing the sequences according to the invention are still suitable as primers, with their Help with the polymerase chain reaction (PCR) DNA of genes which can be produced for O-acetyl homoserine Encode sulfhydrylase.

Such oligonucleotides serving as probes or primers, contain at least 30, preferably at least 20, completely more preferably at least 15 consecutive Nucleotides. Also suitable are oligonucleotides with a length of at least 40 or 50 nucleotides. Optionally, oligonucleotides are also of a length  of at least 100, 250, 200, 250 or 300 nucleotides suitable.

"Isolated" means from its natural environment separated out.

"Polynucleotide" generally refers to Polyribonucleotides and polydeoxyribonucleotides, wherein it unmodified RNA or DNA or modified RNA or DNA can act.

The polynucleotides according to the invention include Polynucleotide according to SEQ ID no. 1 or one of them produced fragment and also those that belong to at least 70%, preferably at least 80% and especially at least 90% to 95% are identical to the Polynucleotide according to SEQ ID no. 1 or one of them produced fragment.

By "polypeptides" is meant peptides or proteins, the two or more linked via peptide bonds Contain amino acids.

The polypeptides of the invention include a polypeptide according to SEQ ID no. 2, in particular those with the biological activity of O-acetyl homoserine sulfhydrylase and also those which are at least 70%, preferably too at least 80%, and especially at least 90% to 95% are identical to the polypeptide according to SEQ ID no. 2 and have the said activity.

The invention further relates to a method for fermentative production of amino acids, in particular L- Lysine and L-methionine, using coryneforms Bacteria, in particular, already have amino acids and at least those for the metY gene amplified nucleotide sequences, in particular be overexpressed.  

The term "reinforcement" describes in this context the increase in intracellular activity of one or of several enzymes in a microorganism that are caused by the corresponding DNA are encoded by, for example the copy number of the gene (s) increases, a strong Promoter used or used for a gene corresponding enzyme with a high activity and coded if necessary, combine these measures.

The microorganisms that are the subject of the present Invention, L-amino acids, in particular L-lysine and L-methionine, from glucose, sucrose, lactose, Fructose, maltose, molasses, starch, cellulose or Glycerol and ethanol. It can be representative coryneformer bacteria especially of the genus Corynebacterium act. In the genus Corynebacterium is in particular the species Corynebacterium glutamicum too which is known in the art for their ability To produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum (C. glutamicum), are especially the known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium molassecola ATCC17965
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
or L-lysine-producing mutants or strains produced therefrom, such as
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 and
Corynebacterium glutamicum DSM5715
or L-methionine-producing mutants or strains produced therefrom, such as
Corynebacterium glutamicum ATCC21608.

The new, for the enzyme O-acetyl homoserine sulfhydrylase (EC 4.2.99.10) was the metY gene of C. glutamicum isolated.

For isolation of the metY gene or other genes of C. glutamicum will initially be a gene bank of this Microorganism in Escherichia coli (E. coli) created. Genbanking is well known Textbooks and manuals written down. When An example is the textbook by Winnacker: Genes and Clones. An Introduction to Gene Technology (Verlag Chemie, Weinheim, Germany, 1990), or the manual of Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A well-known gene bank is that of the E. coli K-12 strain W3110, described by Kohara et al. (Cell 50, 495-508 (1987)) in λ vectors was created. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) describe one Genbank of C. glutamicum ATCC13032, obtained with the help of Cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84: 2160-2164) in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575).

Börmann et al. (Molecular Microbiology 6 (3), 317-326 (1992)) again describe a gene bank of C. glutamicum ATCC13032 using the cosmid pHC79 (scab and Collins, Gene 11, 291-298 (1980)). For the production of a  Genbank of C. glutamicum in E. coli can also use plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) become. Suitable hosts are particularly those E. coli Strains which are restriction and recombination defective. An example of this is the strain DH5αmcr, by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The help of cosmids cloned long DNA fragments can then again in common, for sequencing subcloned suitable vectors and then sequenced be, as it is z. In Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74: 5463-5467, 1977).

The resulting DNA sequences can then be labeled with known Algorithms or sequence analysis programs such. B. the of Staden (Nucleic Acids Research 14, 217-232 (1986)), of Marck (Nucleic Acids Research 16, 1829-1836 (1988)) or Butler's GCG program (Methods of Biochemical Analysis 39, 74-97 (1998)).

The new gene coding for the gene metY DNA sequence of C. glutamicum was found, which is described as SEQ ID no. 1 component of the present invention. Furthermore, from the present DNA sequence with those described above Methods the amino acid sequence of the corresponding protein derived. In SEQ ID no. 2 is the resulting Amino acid sequence of the metY gene product shown.

Coding DNA sequences consisting of SEQ ID no. 1 through the degeneracy of the genetic code are also part of the invention. In the same way are DNA sequences which have SEQ ID NO. 1 or parts of SEQ ID no. 1 hybridize, part of the invention. In the experts are still conservative Amino acid exchanges such. B. Replacement of glycine Alanine or of aspartic acid against glutamic acid in  Proteins known as "sense mutations", which does not lead to a fundamental change in the activity of the Lead protein, d. H. are functionally neutral. Furthermore is known that changes at the N and / or C terminus of a Proteins whose function does not significantly affect or even stabilize. Details can be found on the Expert among others in Ben-Bassat et al. (Journal of Bacteriology 169: 751-757 (1987)), O'Regan et al. (Gene 77: 237-251 (1989)); Sahin-Toth et al. (Protein Sciences 3: 240-247 (1994)); Hochuli et al. (Bio / Technology 6: 1321-1325 (1988)) and in known textbooks of Genetics and molecular biology. Amino acid sequences that correspondingly from SEQ ID no. 2, are also part of the invention.

Similarly, DNA sequences SEQ ID NO. 1 or parts of SEQ ID no. 1 hybridize part of the Invention. Finally, DNA sequences are part of Invention produced by the polymerase chain reaction (PCR) be made using primers that are from SEQ ID no. 1 result. Have such oligonucleotides typically at least 15 nucleotides in length.

Instructions for the identification of DNA sequences by means of Hybridization is found among others in the art Manual "The DIG System Users Guide for Filters Hybridization "the company Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and Liebl et al. (International Journal of Systematic Bacteriology (1991), 41: 255-260). The hybridization takes place under stringent Conditions, that is, only hybrids formed in which probe and target sequence, d. H. the with the Probe treated polynucleotides, at least 70% identical are. It is known that the stringency of Hybridization including washing steps by Varying the buffer composition, the temperature and the Salt concentration is influenced or determined. The  Hybridization reaction is preferably carried out at relative low stringency compared to the washing steps (Hybaid Hybridization Guide, Hybaid Limited, Teddington, UK, 1996).

For the hybridization reaction, for example, a 5 × SSC buffer at a temperature of about 50-68 ° C be used. It can also probe with Hybridize to polynucleotides that are less than 70% Identity to the sequence of the probe. Such hybrids are less stable and are underwent by washing stringent conditions removed. This can be, for example by lowering the salt concentration to 2X SSC and optionally subsequently 0.5 × SSC (The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995) can be achieved, with a Temperature of about 50-68 ° C is set. It is possibly possible the salt concentration up to 0.1 × Lower SSC. By gradually increasing the Hybridization temperature in steps of about 1-2 ° C of 50 to 68 ° C polynucleotide fragments can be isolated for example, at least 70% or at least 80% or at least 90% to 95% identity to the sequence of own used probe. Further instructions for the Hybridization are in the form of so-called kits on the market available (eg DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).

Instructions for the amplification of DNA sequences using the polymerase chain reaction (PCR) will be apparent to those skilled in the art among other things in the handbook of Gait: Oligonukleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and Newton and Graham: PCR (Academic Spectrum Verlag, Heidelberg, Germany, 1994).  

It was found that coryneform bacteria after Overexpression of the metY gene, optionally in combination with the metA gene, in an improved way amino acids, especially L-lysine and L-methionine.

To achieve overexpression, the copy number of the corresponding genes are increased, or it may be the Promoter and regulatory region or the Ribosome binding site located upstream of the Structural gene is mutated. In the same way act expression cassettes upstream of the Structural gene to be installed. By inducible Promoters it is additionally possible to express in the Course of fermentative L-lysine and L-methionine Increase production. By measures for extension The lifetime of mRNA will also be expressed improved. Furthermore, by preventing the degradation the enzyme protein also enhances the enzyme activity. The genes or gene constructs can be either in plasmids with different copy numbers or in the Chromosome be integrated and amplified. alternative may continue to overexpress the genes in question by changing the composition of the media and Cultural guidance can be achieved.

Instructions for this, the expert finds among others Martin et al. (Bio / Technology 5, 137-146 (1987)) Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), to Eikmanns et al. (Gene 102, 93-98 (1991)), in the European Patent 0 472 869, in U.S. Patent 4,601,893 Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)) Remscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in the patent application WO 96/15246 to Malumbres et al. (Gene 134, 15-24 (1993)), Japanese Patent Laid-Open Publication JP-A-10-  229891, by Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), to Makrides (Microbiological Reviews 60: 512-538 (1996)) and in well-known textbooks of genetics and molecular biology.

For amplification, the metY gene according to the invention by way of example with the aid of episomal plasmids overexpressed. Suitable plasmids are those which are known in coryneform bacteria are replicated. numerous known plasmid vectors such. PZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other Plasmid vectors such. For example, those based on pCG4 (US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (US-A 5,158,891) can be used in the same way become.

FIGS. 1 and 2 show examples of such plasmid vectors.

Furthermore, such plasmid vectors are also suitable with the aid of one of the procedure of gene amplification by Integration into the chromosome can apply, just like it for example, Remscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) Duplication or amplification of the hom-thrB operon has been described. This method will do that full gene is cloned into a plasmid vector in a host (typically E. coli), but not in C. can replicate glutamicum. Come as vectors For example, pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994) Journal of Biological Chemistry 269: 32678-84; US-A 5,487,993),  pCR® Blunt (Invitrogen, Groningen, The Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342). The plasmid vector to which the contains amplifying gene is subsequently passed through Conjugation or transformation into the desired strain of C. glutamicum. The method of conjugation For example, Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are for example Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous Recombination by means of a "cross over" event the resulting strain contains at least two copies of the concerned gene.

In addition, it can be used for the production of amino acids, especially L-lysine and L-methionine be beneficial, in addition to the metY gene one or more enzymes of each Biosynthesis, glycolysis, anaplerotic, or To amplify amino acid exports.

For example, for the production of L-lysine, one or more genes selected from the group

• - that for the glyceraldehyde-3-phosphate dehydrogenase coding gene gap (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• - The gene coding for the triose isomerase gene tpi (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),  
• - The gene coding for the 3-phosphoglycerate kinase gene pgk (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• The gene coding for the pyruvate carboxylase pyc (DE-A- 198 31 609),
• - that for a feed back resistant aspartate kinase encoding gene lysC (ACCESSION Number P26512),

amplified, in particular overexpressed.

For example, for the production of L-methionine, one or more genes selected from the group

• - that for the glyceraldehyde-3-phosphate dehydrogenase coding gene gap (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• - The gene coding for the triose isomerase gene tpi (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• - The gene coding for the 3-phosphoglycerate kinase gene pgk (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• The gene coding for the pyruvate carboxylase pyc (DE-A- 198 31 609),
• - that for a feed back resistant aspartate kinase encoding gene lysC (ACCESSION Number P26512),
• The gene coding for the homoserine O-acetyltransferase metA (ACCESSION Number AF052652),
• The gene coding for cystagionin gamma synthase metB (ACCESSION Number AF126953),
• The gene coding for the cystahionin gamma lyase aecD (ACCESSION Number M89931),
• - that coding for the serine hydroxymethyltransferase Gene glyA (JP-A-08107788),

amplified, in particular overexpressed, wherein the additional enhancement of metA is particularly preferred.

Furthermore, it may be advantageous for the production of L-lysine, in addition to the amplification of the metY gene, one or more genes selected from the group

• - The coding for the phosphoenolpyruvate carboxykinase Gene pck (DE 199 50 409.1, DSM 13047),
• - The gene coding for the glucose-6-phosphate isomerase gene pgi (US 09 / 396,478, DSM 12969),
• The gene poxB coding for the pyruvate oxidase (DE: 199 51 975.7; DSM 13114)

attenuate, in particular to reduce expression.

Furthermore, it may be advantageous for the production of L-methionine, in addition to the amplification of the metY gene, one or more genes selected from the group

• - The coding for the phosphoenolpyruvate carboxykinase Gene pck (DE 199 50 409.1, DSM 13047),
• - The gene coding for the glucose-6-phosphate isomerase gene pgi (US 09 / 396,478, DSM 12969),
• The gene poxB coding for the pyruvate oxidase (DE: 199 51 975.7; DSM 13114),
• - The gene coding for homoserine kinase gene thrB (ACCESSION Number P08210),
• - the gene encoding ilvA for threonine dehydratase (ACCESSION Number Q04513),
• The gene encoding threonine synthase, thrC (ACCESSION Number P23669),  
• - that for meso-diaminopimelate D-dehydrogenase coding gene ddh (ACCESSION Number Y00151),

attenuate, in particular to reduce expression.

Furthermore, it can be used for the production of amino acids, especially L-lysine and L-methionine, be beneficial in addition to the overexpression of the metY gene, optionally in combination with the metA-Ken, to eliminate unwanted side reactions (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The microorganisms produced according to the invention can continuous or discontinuous in the batch process (Batch cultivation) or in the fed batch (feed method) or repeated fed batch process (repetitive feed process) for the purpose of producing amino acids, in particular L-lysine and L-methionine are cultured. A Summary of known cultivation methods are in the Textbook by Chmiel (Bioprozesstechnik 1. Introduction to the Bioprocess Engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (Bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

The culture medium to be used must suitably Claims of the respective strains suffice. descriptions of culture media of various microorganisms are in Manual of Methods for General Bacteriology American Society for Bacteriology (Washington D.C., USA, 1981).

As a carbon source, sugars and carbohydrates can z. Glucose, sucrose, lactose, fructose, maltose, Molasses, starch and cellulose, oils and fats such. B. Soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids  such as Palmitic acid, stearic acid and linoleic acid, Alcohols such. As glycerol and ethanol and organic Acids such. As acetic acid can be used. These substances can be used singly or as a mixture.

As a nitrogen source can contain organic nitrogen Compounds such as peptones, yeast extract, meat extract, Malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, Ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate can be used. The nitrogen sources can be used singly or as a mixture.

As a phosphorus source can phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts are used.

As sulfur source, especially for the production Sulfur containing amino acids, organic and inorganic sulfur compounds such as For example, sulfides, sulfites, sulfates and thiosulfates be used.

The culture medium must further contain salts of metals such as As magnesium sulfate or iron sulfate, for the Growth are necessary. Finally, essential Growth substances such as amino acids and vitamins in addition to the above mentioned substances. The culture medium In addition, suitable precursors may be added. The mentioned feedstocks can be used to culture in the form of a added one-off approach or appropriately be fed during cultivation.

For pH control of the culture basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or Ammonia water or acidic compounds such as phosphoric acid or sulfuric acid used in a suitable manner. to Foam processing control can use anti-foaming agents  z. B. fatty acid polyglycol esters are used. to Maintaining the stability of plasmids can the Medium suitable selective substances such. B. Antibiotics are added. To aerobic conditions maintain, become oxygen or oxygen containing gas mixtures such. B. air into the culture entered. The temperature of the culture is usually at 20 ° C to 45 ° C, and preferably at 25 ° C to 40 ° C. The Culture is continued until a maximum of desired product has formed. This goal will be usually within 10 hours to 160 hours reached.

The thus obtained, in particular L-methionine-containing Fermentation broths usually have a dry matter from 7.5 to 25% by weight and contain L-methionine. It is also advantageous if the fermentation at least at the end, but especially over at least 30% The fermentation is driven sugar-limited. The means that during this time the concentration of Recyclable sugar in the fermentation medium to ≧ 0 to 3 g / l held, or lowered.

The produced in this way, in particular L-methionine containing, fermentation broth is then further processed. Depending on the requirement, the biomass wholly or partly by separation methods such. B. the Centrifugation, filtration, decantation or a Combination of this removed from the fermentation broth or completely left in it. Subsequently, will this with known methods such. B. with the help of a Rotary evaporator, thin-film evaporator, Falling film evaporator, by reverse osmosis, or by Nanofiltration thickened or concentrated. This concentrated fermentation broth can subsequently by methods of freeze-drying, spray-drying, spray granulation or by other methods  a preferably free-flowing, finely divided powder be worked up.

This free-flowing, finely divided powder can then turn by suitable compacting or granulation process in a coarse-grained, free-flowing, storable and largely dust-free product are transferred. Advantageous in the Granulation or compaction is the use of usual organic or inorganic auxiliaries, or carriers such as starch, gelatin, Cellulosederivaten or similar substances, such as usually in food or feed processing as a binding, gelling or thickening agent use find, or of other substances such as Silicas, silicates or stearates.

By "free-flowing" is meant powders that consist of a Series of glass pots with different sizes Outlet openings at least from the vessel with the opening 5 mm (Millimeters) without leakage (small, soaps, oils, Fette, Wachse 94, 12 (1968)).

As described here, is with "finely divided", a powder with predominant fraction (<50%) of a grain size of 20 to Meant 200 microns in diameter. With "coarse-grained" are products with a predominant proportion (<50%) of a grain size of 200 meant to 2000 microns in diameter. In this sense means "dust-free" that the product only low levels (<5%) Contains grain sizes smaller than 20 microns in diameter. The Grain size determination can be done with methods of Laser diffraction spectrometry are performed. The appropriate methods are in the textbook for "Particle Size Measurement in Laboratory Practice" by R. H. Müller and R. Schuhmann, Scientific Publishing Company Stuttgart (1996) or in the textbook "Introduction to Particle Technology "by M. Rhodes, Publisher Wiley & Sons (1998) described.  

"Storable", in the sense of this invention, means a Product up to 120 days, preferably up to 52 weeks, particularly preferably 60 months can be stored without a significant loss (<5%) of methionine occurs.

Alternatively, the product can also be applied to one in the Feed processing known and usual organic or inorganic carrier such as Silicas, silicates, shot, bran, flours, starches Sugar or other reared and / or with usual Thickening or binding agents mixed and stabilized become. Application examples and methods for this are in of the literature (The mill + mixed feed technique 132 (1995) 49, page 817).

Finally, the product can by coating method ("Coating") with film formers such as Metal carbonates, silicas, silicates, alginates, Stearates, starches, gums and cellulose ethers, as in DE-C-41 00 920, be brought into a state, in which it is stable against digestion by animal stomachs especially the stomach of ruminants.

If the biomass is separated during the process, be generally more, for example during the Fermentation added inorganic solids removed. In addition, the invention contains Animal feed additive at least the predominant part the dissolved present in the fermentation broth, further formed or added, in particular organic Substances, as far as not by appropriate procedures were separated.

In one aspect of the invention, the biomass may be up to 70%, preferably up to 80%, preferably up to 90%, preferably up to to 95%, and more preferably up to 100% separated become. In a further aspect of the invention, until to 20% of the biomass, preferably up to 15%, preferably up to  10%, preferably up to 5%, more preferably none Biomass separated.

These organic substances include organic By-products of the fermentation optionally used in addition to the L- Methionine produced and optionally excreted. These include L-amino acids selected from the group L- Lysine, L-valine, L-threonine, L-alanine or L-tryptophan. These include vitamins selected from the vitamin B1 group (Thiamine), vitamin B2 (riboflavin), vitamin B5 (Pantothenic acid), vitamin B6 (pyridoxine), vitamin B22 (Cyanocobalamin), nicotinic acid / nicotinic acid amide and vitamin E (tocopherol). These include organic acids, which carry one to three carboxyl groups such as for example Acetic acid, lactic acid, citric acid, malic acid or Fumaric acid. After all, this includes sugar as well Example trehalose. These compounds are optional desired if the nutritional value of the product improve.

These organic substances including L-methionine and / or D-methionine and / or the racemic mixture D, L-methionine can also be given as needed during a suitable process step as a concentrate or Pure substance added in solid or liquid form become. These organic substances can be mentioned individually or as mixtures to the obtained or concentrated Fermentation broth, or even during the drying or Granulation process be added. It is also possible an organic substance or a Mixture of several organic substances to the fermentation broth and another organic substance or another Mixture of several organic substances at a later Process step, for example granulation, add.  

The product described above is used as a feed additive, d. H. Feed additive, suitable for animal nutrition.

The L-methionine content of the animal feed additive is usually 1 wt .-% to 80 wt .-%, preferably 2 wt .-% up to 80% by weight, particularly preferably 4% by weight to 80% by weight, and most preferably 8% to 80% by weight, based on the dry matter of the animal feed additive. Contents of 1 wt .-% to 60 wt .-%, 2 wt .-% to 60 wt .-%, 4% by weight to 60% by weight, 6% by weight to 60% by weight, 1% by weight up to 40% by weight, 2% by weight to 40% by weight or 4% by weight to 40% by weight are also possible. The water content of the Feed additive is usually up to 5 wt .-%, preferably up to 4% by weight, and more preferably less than 2% by weight.

Another object of the invention is accordingly a process for the preparation of an L-methionine-containing animal feed additive from fermentation broths, characterized by the steps

• a) Cultivation and fermentation of an L-methionine producing microorganism in one Fermentation medium;
• b) removal of water from the L-methionine containing Fermentation broth (concentration);
• c) removal of the formed during the fermentation Biomass in an amount of 0 to 100% by weight; and
• d) drying the obtained according to a) and / or b) Fermentation broth to the animal feed additive in to obtain the desired powder or granular form.

If desired, one or more of the following steps may be carried out in the process according to the invention:

• a) addition of one or more of the organic substances, including L-methionine and / or D-methionine and / or the racemic mixture D, L-methionine the products obtained according to a), b) and / or c);
• b) addition of auxiliaries to those obtained according to a) to d) Substances to stabilize and increase the Shelf life selected from the group silicic acids, Silicates, stearates, shot and bran; or
• c) conversion of the substances obtained according to a) to e) into an animal stomach in particular rumen stable form through Coating with film formers.

The analysis of L-lysine and L-methionine can be done by Ion exchange chromatography with subsequent ninhydrin Derivatization done, as in Spackman et al. (Analytical Chemistry, 30, (1958), 1190).

The following microorganism was deposited as a pure culture on 13. 05. 00 at the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the Budapest Treaty:

• Corynebacterium glutamicum strain DSM5715 / pCREmetY as DSM 13556

The inventive method is used for fermentative Production of amino acids, in particular L-lysine and L-lysine Methionine.  

Examples

The present invention will be described below with reference to Embodiments explained in more detail.

example 1 Preparation of a Genomic Cosmid Gene Bank Corynebacterium glutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was as in Tauch et al. (1995, Plasmid 33: 168-179) described isolated and with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product description Sau3AI, Code no. 27-0913-02) partial split. The DNA fragments were shrimp with alkaline Phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product description SAP, code no. 1758250) dephosphorylated. The DNA of the cosmid vector SuperCos1 (Wahl et al., 1987, Proceedings of the National Academy of Sciences, USA, 84: 2160-2164), obtained from the company Stratagene (La Jolla, USA, Product description SuperCos1 Cosmid Vector Kit, code no. 251301) was used with the Restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Code no. 27-0948-02) cleaved and also with shrimp alkaline phosphatase dephosphorylated.

Subsequently, the cosmid DNA with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). The cosmid DNA treated in this way was labeled with the treated ATCC13032 DNA and the mixture with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4 DNA Ligase, Code no. 27-0870-04) treated. The ligation mixture was then washed with Help with the Gigapack II XL Packing Extract (Stratagene, La  Jolla, USA, Product description Gigapack II XL Packing Extract, Code no. 200217) in phages.

To infect the E. coli strain NM554 (Raleigh et al 1988, Nucleic Acid Research 16: 1563-1575), the cells were taken up in 10 mM MgSO ₄ and mixed with an aliquot of the phage suspension. Infection and titering of the cosmidbank were performed as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), where the cells were plated on LB agar (Lennox, 1955, Virology, 1: 190) with 100 mg / L ampicillin. After incubation overnight at 37 ° C, recombinant single clones were selected.

Example 2 Isolation and sequencing of the metY gene

The cosmid DNA of a single colony was treated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) isolated according to the manufacturer and with the Restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, product no. 27- 0913-02) partially split. The DNA fragments were with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product no. 1758250) dephosphorylated. After gel electrophoresis Separation was the isolation of the cosmid fragments in Size range from 1500 to 2000 bp with the QiaExII gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

The DNA of the sequencing vector pZero-1 obtained from the company Invitrogen (Groningen, Netherlands, product description Zero Background Cloning Kit, Product No. K2500-01) with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04). The ligation of  Cosmidfragmente in the sequencing vector pZero-1 was like by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), wherein the DNA mixture with T4 ligase (Pharmacia Biotech, Freiburg, Germany) was incubated overnight. This Ligation mixture was then added to the E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences, U.S.A., 87: 4645-4649) (Tauch et al. 1994, FEMS Microbiol. Letters, 123: 343-7) and on LB Agar (Lennox, 1955, Virology, 1: 190) with 50 mg / L zeocin plated.

The plasmid preparation of the recombinant clones was carried out with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). The sequencing was carried out after the dideoxy Chain termination method of Sanger et al. (1977, Proceedings of the National Academy of Sciences U.S.A., 74: 5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067). It became the "RR dRhodamin Terminator Cycle Sequencing Kit "by PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany). Gel electrophoretic separation and analysis of the Sequencing reaction was carried out in a "rotiphoresis NF Acrylamide / Bisacrylamide "Gel (29: 1) (Product No. A124.1, Roth, Karlsruhe, Germany) with the "ABI Prism 377" Sequencer from PE Applied Biosystems (Weiterstadt, Germany).

The obtained crude sequence data were then under Application of the Staden Program Package (1986, Nucleic Acids Research, 14: 217-231), version 97-0. The Single sequences of pZero1 derivatives became one coherent contig assembled. The Computerized coding area analysis was conducted with the Program XNIP (Staden, 1986, Nucleic Acids Research, 14: 217-231).  

The nucleotide sequence obtained is shown in SEQ ID no. 1 shown. Analysis of the nucleotide sequence revealed open reading frame of 1313 base pairs, which is called metY Gene was designated. The metY gene encodes a protein of 437 amino acids.

Example 3 Construction of vectors for the expression of metY and metAY 3.1. Amplification of the genes metY and metA

The methionine biosynthesis genes metA and metY from C. glutamicum were obtained using the polymerase Chain reaction (PCR) and synthetic oligonucleotides amplified. Starting from the nucleotide sequences of Methionine biosynthesis genes metY (SEQ ID No. 1) and metA (Genbank entry Accession Number AF052652) of C. glutamicum ATCC 13032 PCR primers were synthesized (MWG Biotech, Ebersberg, Germany). These primers were so selected that the amplified fragments the genes as well their native ribosome binding sites, but not possible Promoter regions included. In addition, suitable Restriction interfaces inserted, which cloning in enable the target vector. The sequences of the PCR primers, the inserted interfaces (sequence underlined) and the amplified gene (fragment size, in bp, is in Brackets listed) are in the following Table 1 listed.  

Table 1

The PCR experiments were carried out with the Taq DNA polymerase of Gibco-BRL (Eggestein, Germany) in a "PCT-100 Thermal Cycler "(MJ Research Inc., Watertown, Mass., USA) carried out. On a single denaturation step followed by a denaturation step of 2 minutes at 94 ° C 90 seconds (sec) at 94 ° C, one annealing step for 90 sec at a primer-dependent temperature of T = (2 × AT + 4 × GC) - 5C (Suggs, et al., 1981, pp. 683-693, In: D.D. Brown, et al Fox, C. Eds., Developmental Biology using Purified Genes. Academic Press, New York, USA) and a 90 sec continuous extension step at 72 ° C. The last three Steps were repeated cyclically 35 times and the reaction was with a final extension step of 10 minutes (min) ended at 72 ° C. The products thus amplified were electrophoresed in a 0.8% agarose gel checked.  

The 1341 bp metY fragment was used with the Restriction endonucleases SalI and NsiI cleaved, 1161 bp large metA fragment with the restriction endonucleases EcoRI and BamHI. Both approaches were gel electrophoretically separated and the fragments metY (about 1330 bp) and metA (about 1150 bp) from the agarose gel with the QiaExII gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) isolated.

3.2. Cloning of metY into the vector pZ8-1

As a base vector for expression in both C. glutamicum and also in E. coli was the E. coli - C. glutamicum shuttle - Expression vector pZ8-1 (EP 0 375 889) used. DNA This plasmid was treated with the restriction enzymes SalI and PstI completely split and then shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) dephosphorylated. That in example 3.1 from the agarose gel isolated metY fragment was prepared with the so Vector pZ8-1 mixed and the batch with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4 DNA Ligase, Code no. 27-0870-04) treated.

The ligation mixture was transformed into E. coli strain DH5α (Hanahan, In: DNA Cloning, A Practical Approach, Vol I.IRL Press, Oxford, Washington DC, USA). The selection of plasmid-carrying cells was carried out by plating out the transformation mixture on LB agar (Lennox, 1955, Virology, 1: 190) with 50 mg / l kanamycin. After incubation overnight at 37 ° C, recombinant single clones were selected. Plasmid DNA was isolated from a transformant using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and checked by restriction cleavage. The resulting plasmid was named pCREmetY. It is shown in Fig. 1.

3.3. Cloning of metA and metY into the vector pZ8-1

DNA of plasmid pZ8-1 was mixed with the restriction enzymes EcoRI and BamHI completely cleaved and then with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product no. 1758250) dephosphorylated. That in example 3.1 from the Agarose gel isolated metA fragment was made with the so prepared vector pZ8-1 mixed and the batch with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4 DNA Ligase, Code no. 27-0870-04) treated.

The ligation mixture was transformed into E. coli strain DH5α (Hanahan, In: DNA Cloning, A Practical Approach, Vol. IRL-Press, Oxford, Washington DC, USA). The Selection of plasmid-carrying cells was carried out by Plating the transformation mixture on LB agar (Lennox, 1955, Virology, 1: 190) with 50 mg / l kanamycin. After incubation overnight at 37 ° C, recombinant Selected individual clones. Plasmid DNA was from a Transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to manufacturer's instructions isolated and checked by restriction cleavage. The obtained plasmid was called pCREmetA.

The plasmid pCREmetA was digested with the restriction enzymes SalI and PstI completely split and then shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) dephosphorylated. That in example 3.1 from the agarose gel isolated metY fragment was prepared with the so Vector pCREmetA mixed and the batch with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4 DNA Ligase, Code no. 27-0870-04) treated.  

The ligation mixture was transformed into E. coli strain DH5α (Hanahan, In: DNA cloning, A Practical Approach, Vol I.IRL Press, Oxford, Washington DC, USA). The selection of plasmid-carrying cells was carried out by plating out the transformation mixture on LB agar (Lennox, 1955, Virology, 1: 190) with 50 mg / l kanamycin. After incubation overnight at 37 ° C, recombinant single clones were selected. Plasmid DNA was isolated from a transformant using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and checked by restriction cleavage. The resulting plasmid was named pCREmetAY. It is shown in FIG. 2.

Example 4 Preparation of strains DSM5715 / pCREmetY and DSM5715 / pCREmetAY

The vectors pCREmetY and pCREmetAY mentioned in Examples 3.2 and 3.3 were analyzed by the electroporation method of Tauch et al. (1994, FEMS Microbiological Letters, 123: 343-347) electroporated into Corynebacterium glutamicum DSM 5715. The strain DSM 5715 is an AEC-resistant lysine producer. The selection of plasmid-carrying cells was made by plating out the electroporation batch on LB agar (Sambrook et al, Molecular Cloning:... A Laboratory Manual 2 ^nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), the supplemented with 25 mg / l kanamycin. Plasmid DNA was isolated from one transformant by the usual methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927) and checked by restriction digestion followed by agarose gel electrophoresis. The strains were named DSM5715 / pCREmetY and DSM5715pCREmetAY. The strain DSM5715 / pCREmetY was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the terms of the Budapest Treaty as DSM 13556.

Example 5 Preparation of lysine with the strain DSM5715 / pCREmetY

The C. glutamicum strain obtained in Example 4 DSM5715 / pCREmetY was in production for lysine cultivated suitable nutrient medium and the lysine content in the Culture supernatant determined.

For this, the strain was first on agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (50 mg / l)) for 24 hours at 33 ° C. outgoing from this agar plate culture, a preculture was inoculated (10 ml of medium in 100 ml Erlenmeyer flask). As a medium for the preculture was used the complete medium CgIII.

Medium Cg III

NaCl 2.5 g / l Bacto-peptone 10 g / l Bacto-yeast extract 10 g / l Glucose (separately autoclaved) 2% (w / v) AL = L <The pH was adjusted to pH 7.4

To this was added kanamycin (50 mg / L). The preculture was 16 hours at 33 ° C at 240 rpm on the shaker incubated. From this Vorkultur became a main culture seeded so that the initial OD (660 nm) of the major culture 0.1 was. For the main culture, the medium MM used.  

Medium MM

CSL (Corn Steep Liquor) 5 g / l MOPS (morpholinopropanesulfonic acid) 20 g / l Glucose (separately autoclaved) 50 g / l (NH ₄ ) ₂ SO ₄ 25 g / l KH ₂ PO ₄ 0.1 g / l MgSO ₄ .7H ₂ O 1.0 g / l CaCl ₂ .2H ₂ O 10 mg / l FeSO ₄ .7H ₂ O 10 mg / l MnSO ₄ .H ₂ O 5.0 mg / l Biotin (sterile filtered) 0.3 mg / l Thiamine.HCl (sterile filtered) 0.2 mg / l L-leucine (sterile filtered) 0.1 g / l CaCO ₃ 25 g / l

CSL, MOPS and saline were adjusted to pH 7 with ammonia water and autoclaved. Subsequently, the sterile substrate and vitamin solutions were added, as well as the dry autoclaved CaCO ₃ .

The cultivation is carried out in 10 ml volume in a 100 ml Erlenmeyer flask with harassment. It became kanamycin (50 mg / l) added. The cultivation was carried out at 33 ° C and 80% humidity.

After 48 hours, the OD at a measuring wavelength of 660 nm with the Biomek 1000 (Beckmann Instruments GmbH,  Munich). The amount of lysine formed was with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection certainly.

Table 2 shows the result of the experiment.

Table 2

Example 6 Preparation of methionine with strain DSM5715 / pCREmetAY

The C. glutamicum strain obtained in Example 4 DSM5715 / pCREmetAY was in production for one of Methionine cultivated suitable nutrient medium and the Methionine content determined in the culture supernatant.

For this, the strain was first on agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (50 mg / l)) for 24 hours at 33 ° C. outgoing from this agar plate culture, a preculture would be inoculated (10 ml of medium in 100 ml Erlenmeyer flask). As a medium for the preculture became the complete medium CgIII, as in example 5 described, used.

To this was added kanamycin (50 mg / L). The preculture was 16 hours at 33 ° C at 290 rpm on the shaker incubated. From this Vorkultur became a main culture seeded so that the initial OD (660 nm) of the major culture  0.1 was. For the main culture, the medium MM was as in Example 5 described used.

CSL, MOPS and saline were adjusted to pH 7 with ammonia water and autoclaved. Subsequently, the sterile substrate and vitamin solutions were added, as well as the dry autoclaved CaCO ₃ .

The cultivation is carried out in 10 ml volume in a 100 ml Erlenmeyer flask with harassment. It became kanamycin (50 mg / l) added. The cultivation was carried out at 33 ° C and 80% humidity.

After 72 hours, the OD became at a measuring wavelength of 660 nm with the Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of methionine formed was with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection certainly.

Table 3 shows the result of the experiment.

Table 3

The following figures are attached:

• - Fig. 1: Plasmid pCREmetY
• Fig. 2: Plasmid pCREmetAY

The abbreviations used in the figures have the following meaning:
Kan: resistance gene for kanamycin
metY: metY gene of C. glutamicum
metA: metA gene of C. glutamicum
Ptac: tac promoter
rrnB-T1T2: terminator T1T2 of the E. coli rrnB gene
rep: plasmid-encoded replication origin for C. glutamicum (from pHM1519)
BamHI: Interface of the restriction enzyme BamHI
EcoRI: restriction enzyme EcoRI site
EcoRV: restriction enzyme EcoRV site
PstI: restriction enzyme PstI site
SalI: Site of the restriction enzyme SalI
XhoI: Interface of the restriction enzyme XhoI

SEQUENCE LISTING

Claims (36)

An isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence encoding the metY gene selected from the group

• a) polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO. 2 contains
• b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO. 2,
• c) polynucleotide which is complementary to the polynucleotides of a) or b), and
• d) polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of a), b) or c),

wherein the polypeptide preferably has the activity of O-acetyl homoserine sulfhydrylase. 2. Polynucleotide according to claim 1, wherein the Polynucleotide one in coryneform bacteria is replicable, preferably recombinant DNA. 3. Polynucleotide according to claim 1, wherein the Polynucleotide is an RNA. 4. Polynucleotide according to claim 2 or 3, containing the Nucleic acid sequence as in SEQ ID no. 1 shown. 5. A replicatable DNA according to claim 2 or 3, comprising

• a) the nucleotide sequence shown in SEQ ID no. 1, or
• b) at least one sequence corresponding to the sequence (i) within the range of the degeneracy of the genetic code, or
• c) at least one sequence that hybridizes with the sequence complementary to sequence (i) or (ii), and optionally
• d) functionally neutral sense mutations in (i).

6. A replicable DNA according to claim 5, characterized characterized in that the hybridization corresponding to the sequence (iii) under a stringency at most 2 × SSC is performed. 7. polynucleotide sequence according to claim 2 or 3, which for encodes a polypeptide having the amino acid sequence in SEQ ID no. 2 represents. 8. A process for the fermentative production of L-amino acids, in particular L-lysine, characterized in that one carries out the following steps:

• a) fermentation of the desired amino acid-producing coryneform bacteria, in which at least the metY gene or nucleotide sequences coding therefor are enhanced, in particular overexpressed;
• b) accumulation of the L-amino acid in the medium or in the cells of the bacteria; and
• c) isolate from the L-amino acid.

9. A process for the fermentative production of L-amino acids, in particular L-methionine, characterized in that one carries out the following steps:

• a) fermentation of the L-methionine-producing coryneform bacteria, in which one optionally amplifies the gene metY with metA, in particular overexpressed;
• b) accumulation of the L-amino acid in the medium or in the cells of the bacteria; and
• c) isolate from the L-amino acid.

10. The method according to claim 8 or 9, characterized characterized in that you have bacteria used, in which additional genes of the Biosyntheseweges the desired L-amino acid strengthened. 11. The method according to claim 8 or 9, characterized characterized in that you have bacteria in which the metabolic pathways at least are partially switched off, which is the formation of the reduce the desired amino acid. 12. The method according to claim 8, characterized characterized in that one with a Plasmid vector transformed strain used, and the Plasmid vector the metY gene and optionally additionally carries the metA gene. 13. The method according to claim 9, characterized characterized in that one with a Plasmid vector transformed strain used, and the Plasmid vector representing the genes metA and metY carries coding nucleotide sequence. 14. The method according to claim 8, characterized in that one fermented for the production of L-amino acids, in particular L-lysine, coryneform microorganisms in which one or more of the genes selected from the group

• 1. 14.1 the gene gap coding for the glyceraldehyde-3-phosphate dehydrogenase,
• 2. 14.2 the gene tpi coding for the triose phosphate isomerase,
• 3. 14.3 the gene pgk coding for the 3-phosphoglycerate kinase,
• 4. 14.4 the gene coding for pyruvate carboxylase pyc,
• 5. 14.5 the lysC gene encoding a feedback-resistant aspartate kinase

amplified, in particular overexpressed. 15. The method according to claim 9, characterized in that one fermented for the production of L-amino acids, in particular L-methionine, coryneform microorganisms in which one or more of the genes selected from the group

• 1. 15.1 the lysC gene coding for a feedback-resistant aspartate kinase,
• 2. 15.2 the gene gap coding for the glyceraldehyde-3-phosphate dehydrogenase,
• 3. 15.3 the gene tpi coding for the triosephosphate isomerase,
• 4. 15.4 the gene metA coding for the homoserine O-acetyltransferase,
• 5. 15.5 the gene encoding cystahionine gamma synthase metB,
• 6. 15.6 the gene coding for the cystahionine gamma lyase aecD,
• 7. 15.7 the gene glyA coding for serine hydroxymethyltransferase,
• 8. 15.8 the gene pgk coding for the 3-phosphoglycerate kinase,
• 9. 15.9 the gene coding for pyruvate carboxylase pyc

amplified, in particular overexpressed. 16. The method according to claim 15, characterized characterized in that for the production of L-amino acids, in particular L-methionine, fermented coryneform microorganisms containing a additional amplification of the metY gene by metA respectively. 17. The method according to claim 8, characterized in that fermented for the production of L-amino acids, in particular L-lysine, coryneform microorganisms which an additional amplification of the metY gene by attenuation, in particular reduction of the expression of one or more genes selected from the group

• 1. 17.1 the phosphoenolpyruvate carboxykinase-encoding gene pck
• 2. 17.2 the gene coding for the glucose-6-phosphate isomerase gene pgi
• 3. 17.3 the pyruvate oxidase-encoding gene poxB.

18. The method according to claim 9, characterized in that one fermented for the production of L-amino acids, in particular L-methionine, coryneform microorganisms in which one or more of the genes selected from the group

• 1. 18.1 the homoserine kinase encoding gene thrB
• 2. 18.2 the gene encoding ilvA for the threonine dehydratase
• 3. 18.3 the threonine synthase-encoding gene thrC
• 4. 18.4 the gene coding for meso-diaminopimelate D-dehydrogenase ddh
• 5. 18.5 the phosphoenolpyruvate carboxykinase encoding gene pck
• 6. 18.6 the gene coding for the glucose-6-phosphate isomerase gene pgi
• 7. 18.7 the gene coding for pyruvate oxidase poxE

weakens or reduces the expression. 19. Coryneform bacteria in which the metY gene amplified, in particular overexpressed. 20. Coryneform bacteria containing a vector that a polynucleotide according to claim 1 carries. 21. Method according to one or more of the preceding Claims, characterized in that one Microorganisms of the species Corynebacterium glutamicum starts. 22. A process for the preparation of an L-methionine-containing animal feed additive from fermentation broths, characterized by the steps:

• a) culturing and fermenting a L-methionine producing microorganism in a fermentation medium;
• b) removal of water from the L-methionine-containing fermentation broth (concentration);
• c) removing the biomass formed during the fermentation in an amount of 0 to 100 wt .-%; and
• d) drying the fermentation broth obtained according to b) and / or c) in order to obtain the animal feed additive in the desired powder or granule form.

23. Method according to claim 22, characterized in that that one uses microorganisms in which one additional genes of the biosynthetic pathway of L- Reinforced methionine. 24. The method according to claim 23, characterized characterized in that one microorganisms in which the metabolic pathways at least are partially switched off, which is the formation of Reduce methionine. 25. The method according to claim 23, characterized characterized in that the expression the polynucleotides encoding the metY gene amplified, in particular overexpressed. 26. Method according to one or more of the preceding Claims 22-25, characterized characterized in that one microorganisms of the species Corynebacterium glutamicum. 27. The method according to claim 22, characterized in that one additionally performs one or more of the following steps:

• a) addition of one or more of the organic substances, including L-methionine and / or D-methionine and / or the racemic mixture D, L-methionine, to the products obtained according to b), c) and / or d);
• b) addition of auxiliaries to the substances obtained according to b) to e) for stabilizing and increasing the shelf life selected from the group silicas, silicates, stearates, shot and bran; or
• c) transfer of the substances obtained according to b) to f) into an animal stomach, in particular rumen stable form by coating with film formers.

28. The method according to claim 22 or 27, characterized characterized in that a part of the biomass is removed becomes. 29. The method according to claim 28, characterized that up to 100% of the biomass is removed. 30. The method according to claim 22 or 27, characterized characterized in that the water content is up to 5 wt .-% is. 31. The method according to claim 30, characterized the water content is less than 2% by weight. 32. The method according to claim 27, 28, 29, 30 or 31, characterized in that the film former Metal carbonates, silicas, silicates, alginates, Stearates, starches, gums or cellulose ethers are. 33. Animal feed additive prepared according to claims 22 to 32. 34. animal feed additive according to claim 33, characterized characterized in that it contains 1% by weight to 80% by weight Methionine, D-methionine, D, L-methionine or one Mixture thereof, based on the dry matter of the Animal Feed Additive.   35. Method for finding RNA, cDNA and DNA to Nucleic acids, or polynucleotides or Isolating genes responsible for O-acetyl homoserine Sulfhydrolase encode or a high similarity with the sequence of the metY gene thereby characterized in that the Polynucleotide containing the polynucleotide sequences according to claims 1, 2, 3 or 4 as Hybridization probes used. 36. Corynebacterium glutamicum strain DM5715 / pCREmetY deposited with the DSMZ, Braunschweig, under the no. DSM 12840.